Recent evidence from our laboratories and others has identified a cathepsin B-like cysteine proteinase (CB) as an important enzyme in the processes of tumor invasion and metastasis. CB from liver has been shown to degrade collagen and proteoglycans (components of the extracellular matrix) and to activate latent collagenase. Thus CB may facilitate tumor metastasis by enabling the tumor cell to invade at primary and metastatic sites and to intravasate and extravasate. CB is released from tumors apparently as a proenzyme which has increased stability. We will purify CB from subcutaneous tumors of the murine B16 amelanotic melanoma (B16a) and from B16a - conditioned cultured media. These enzymes will be characterized for their physical & kinetic properties. Proteolytic activation of proCB will be assessed using active CB, plasmin, plasminogen activator, kallikrein, etc. Monoclonal antibodies to active CB and to proCB will be developed, and their cross-reactivities tested against one another as well as against CB purified from other tumors and other tissues. Using purified CB we will determine its ability to degrade, in the presence and absence of monoclonal antibodies, components of the extracellular matrix [collagen (types I-V), laminin, fibronectin]. The ability of CB to activate tumor-derived latent type IV collagenase will also be determined. In vitro assays to study invasion of tumor cells throught an endothelial monolayer and through amnion-derived basement membrane will be established. Invasion by 125I-Udr labeled tumor cells (B16a and M5076 reticulum cell sarcoma) will be followed in the presence and absence of monoclonal antibodies to CB. These studies should enable us to assess the presumptive role of CB in tumor cell metastasis and to assess whether CB may be one proteolytic enzyme in a metastatic proteolytic cascade. For example, plasmin derived from the action of plasminogen activator (PA) on plasminogen may activate proCB and CB in turn may activate latent type IV collagenase. It might be possible to interrupt such a proteolytic cascade by directing therapeutic interventions towards preventing release of proCB or towards the pro fragment of proCB (since proCB is not released from normal cells).